We have studied the post translational modifications of the insulin receptor, i.e., glycosylation, fatty acylation, and phosphorylation using biosynthesis labeling of the insulin receptor. After labeling the insulin receptor with radioactive sugars, the individual subunits were isolated and tryptic peptides made from each subunit. These peptides were separated using high performance liquid chromatography and the glycosylated peptides detected by counting the fractions in a liquid scintillation counter. Based on the differential sensitivity of these labeled peptides to peptide: N-glycosidase F, an enzyme that removes high mannose and complex N-linked oligosaccharides, and endo-a-N-acetylgalactosaminindase, an enzyme that removes O-linked oligosaccharides, we have shown that the insulin receptor contains O-linked oligosaccharides. These 0-linked carbohydrates are on the beta subunit. The peptide containing the 0-linked oliogosaccharides was localized -to the amino terminus of the beta subunit. An anti-peptide antibody made to the 12 amino acids at the amino terminus of the beta subunit was able to immunoprecipitate specifically the tryptic fragment containing the N- and O-linked oligosaccharides. Using an inhibitor of O-linked glycosylation, we investigated the function of the O-linked glycosylation. In the presence of this inhibitor the binding of insulin to the insulin receptor was unchanged.